Gene therapy 

is the insertion, alteration, or removal of geneswithin an individual's cells and biological

tissues to treat disease. It is a technique for correcting defective genes that are responsible for disease
development.[1] The most common form of gene therapy involves the insertion of functional genes into an
unspecified genomic location in order to replace a mutated gene, but other forms involve directly
correcting the mutation or modifying normal gene that enables a viral infection. Although the technology is
still in its infancy, it has been used with some success.

Contents
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[ edit]Approach
Scientists have taken the logical step of trying to introduce genes directly into human cells, focusing on
diseases caused by single-gene defects, such as cystic fibrosis, haemophilia, muscular
dystrophy and sickle cell anemia. However, this has proven more difficult than modifying bacteria,
primarily because of the problems involved in carrying large sections of DNA and delivering them to the
correct site on the gene. Today, most gene therapy studies are aimed at cancer and hereditary diseases
linked to a genetic defect. Antisense therapy is not strictly a form of gene therapy, but is a related,
genetically-mediated therapy.

The most common form of genetic engineering involves the insertion of a functional gene at an
unspecified location in the host genome.This is accomplished by isolating and copying the gene of
interest, generating a construct containing all the genetic elements for correct expression, and then
inserting this construct into a random location in the host organism. Other forms of genetic engineering
include gene targeting and knocking out specific genes via engineered nucleases such as zinc
finger nucleases, engineered I-CreI homing endonucleases, or nucleases generated from TAL
effectors. An example of gene-knockout mediated gene therapy is the knockout of the
human CCR5 gene in T-cells in order to control HIV infection.[2] This approach is currently being used
in several human clinical trials.[3]

The biology of human gene therapy remains complex and many techniques need further development.
Many diseases and their strict genetic link need to be understood more fully before gene therapy can be
used appropriately. The public policy debate surrounding the possible use of genetically engineered
material in human subjects has been equally complex. Major participants in the debate have come from
the fields of biology, government, law, medicine, philosophy, politics, and religion, each bringing different
views to the discussion.[citation needed]

[ edit]Types of gene therapy

Gene therapy may be classified into the two following types:

[edit]Germ line gene therapy

In the case of germ line gene therapy, germ cells, i.e., sperm or eggs, are modified by the introduction of
functional genes, which are integrated into their genomes. Therefore, the change due to therapy would be
heritable and would be passed on to later generations. This new approach, theoretically, should be highly
effective in counteracting genetic disorders and hereditary diseases. However, many jurisdictions prohibit
this for application in human beings, at least for the present, for a variety of technical and ethical reasons.
[citation needed]

[edit]Somatic gene therapy

In the case of somatic gene therapy, the therapeutic genes are transferred into the somatic cells of a
patient. Any modifications and effects will be restricted to the individual patient only, and will not be
inherited by the patient's offspring or later generations.

[ edit]Vectors in gene therapy

[edit]Viruses
Main article:  Viral vector

All viruses bind to their hosts and introduce their genetic material into the host cell as part of their
replication cycle. This genetic material contains basic 'instructions' of how to produce more copies of
these viruses, hijacking the body's normal production machinery to serve the needs of the virus. The host
cell will carry out these instructions and produce additional copies of the virus, leading to more and more
cells becoming infected. Some types of viruses insert their genes into the host's genome, but do not
actually enter the cell. Others penetrate the cell membrane disguised as protein molecules and enter the
cell.

There are two main types of virus infection: lytic and lysogenic. Shortly after inserting its DNA, viruses
of the lytic cycle quickly produce more viruses, burst from the cell and infect more cells. Lysogenic viruses
integrate their DNA into the DNA of the host cell and may live in the body for many years before
responding to a trigger. The virus reproduces as the cell does and does not inflict bodily harm until it is
triggered. The trigger releases the DNA from that of the host and employs it to create new viruses. HIV is
a lysogenic infection. Some scientists believe that if they find the origin of its trigger, they will be able to
stop the virus from ever reproducing throughout the body.[citation needed]

[edit]Retroviruses

The genetic material in retroviruses is in the form of RNA molecules, while the genetic material of their
hosts is in the form of DNA. When a retrovirus infects a host cell, it will introduce its RNA together with
some enzymes, namely reverse transcriptase and integrase, into the cell. This RNA molecule from the
retrovirus must produce a DNA copy from its RNA molecule before it can be integrated into the genetic
material of the host cell. The process of producing a DNA copy from an RNA molecule is termed reverse
transcription. It is carried out by one of the enzymes carried in the virus, called reverse
transcriptase. After this DNA copy is produced and is free in the nucleus of the host cell, it must be
incorporated into the genome of the host cell. That is, it must be inserted into the large DNA molecules in
the cell (the chromosomes). This process is done by another enzyme carried in the virus
called integrase.

Now that the genetic material of the virus has been inserted, it can be said that the host cell has been
modified to contain new genes. If this host cell divides later, its descendants will all contain the new
genes. Sometimes the genes of the retrovirus do not express their information immediately.

One of the problems of gene therapy using retroviruses is that the integrase enzyme can insert the
genetic material of the virus into any arbitrary position in the genome of the host; it randomly inserts the
genetic material into a chromosome. If genetic material happens to be inserted in the middle of one of the
original genes of the host cell, this gene will be disrupted (insertional mutagenesis). If the gene
happens to be one regulating cell division, uncontrolled cell division (i.e., cancer) can occur. This
problem has recently begun to be addressed by utilizingzinc finger nucleases[4] or by including certain
sequences such as the beta-globin locus control region to direct the site of integration to specific
chromosomal sites.

Gene therapy trials using retroviral vectors to treat X-linked severe combined

immunodeficiency (X-SCID) represent the most successful application of gene therapy to date. More
than twenty patients have been treated in France and Britain, with a high rate of immune system
reconstitution observed. Similar trials were restricted or halted in the USA when leukemia was reported in
patients treated in the French X-SCID gene therapy trial.[citation needed] To date, four children in the French
trial and one in the British trial have developed leukemia as a result of insertional mutagenesis by the
retroviral vector. All but one of these children responded well to conventional anti-leukemia treatment.
Gene therapy trials to treat SCID due to deficiency of the Adenosine Deaminase (ADA) enzyme continue
with relative success in the USA, Britain, Ireland, Italy and Japan.
[edit]Adenoviruses

Adenoviruses are viruses that carry their genetic material in the form of double-stranded DNA. They
cause respiratory, intestinal, and eye infections in humans (especially the common cold). When these
viruses infect a host cell, they introduce their DNA molecule into the host. The genetic material of the
adenoviruses is not incorporated (transient) into the host cell's genetic material. The DNA molecule is left
free in the nucleus of the host cell, and the instructions in this extra DNA molecule are transcribed just
like any other gene. The only difference is that these extra genes are not replicated when the cell is about
to undergo cell division so the descendants of that cell will not have the extra gene. As a result, treatment
with the adenovirus will require readministration in a growing cell population although the absence of
integration into the host cell's genome should prevent the type of cancer seen in the SCID trials. This
vector system has been promoted for treating cancer and indeed the first gene therapy product to be
licensed to treat cancer, Gendicine, is an adenovirus. Gendicine, an adenoviral p53-based gene
therapy was approved by the Chinese FDA in 2003 for treatment of head and neck cancer. Advexin, a
similar gene therapy approach from Introgen, was turned down by the US FDA in 2008.

Concerns about the safety of adenovirus vectors were raised after the 1999 death of Jesse
Gelsinger while participating in a gene therapy trial. Since then, work using adenovirus vectors has
focused on genetically crippled versions of the virus.
[edit]Adeno-associated viruses

Adeno-associated viruses, from the parvovirus family, are small viruses with a genome of single
stranded DNA. The wild type AAV can insert genetic material at a specific site on chromosome 19 with
near 100% certainty. But the recombinant AAV, which does not contain any viral genes and only the
therapeutic gene, does not integrate into the genome. Instead the recombinant viral genome fuses at its
ends via the ITR (inverted terminal repeats) recombination to form circular, episomal forms which are
predicted to be the primary cause of the long term gene expression. There are a few disadvantages to
using AAV, including the small amount of DNA it can carry (low capacity) and the difficulty in producing it.
The production problem however has recently been solved by Amsterdam Molecular Therapeutics.
[5]
 This type of virus is being used, however, because it is non-pathogenic (most people carry this
harmless virus). In contrast to adenoviruses, most people treated with AAV will not build an immune
response to remove the virus and the cells that have been successfully treated with it. Several trials with
AAV are on-going or in preparation, mainly trying to treat muscle and eye diseases; the two tissues where
the virus seems particularly useful. However, clinical trials have also been initiated where AAV vectors are
used to deliver genes to the brain. This is possible because AAV viruses can infect non-dividing
(quiescent) cells, such as neurons in which their genomes are expressed for a long time.
[edit]Envelope protein pseudotyping of viral vectors

The viral vectors described above have natural host cell populations that they infect most
efficiently. Retroviruses have limited natural host cell ranges, and although adenovirus and adeno-
associated virus are able to infect a relatively broader range of cells efficiently, some cell types are
refractory to infection by these viruses as well. Attachment to and entry into a susceptible cell is mediated
by the protein envelope on the surface of a virus. Retroviruses and adeno-associated viruses have a
single protein coating their membrane, while adenoviruses are coated with both an envelope protein and
fibers that extend away from the surface of the virus. The envelope proteins on each of these viruses
bind tocell-surface molecules such as heparin sulfate, which localizes them upon the surface of
the potential host, as well as with the specificprotein receptor that either induces entry-promoting
structural changes in the viral protein, or localizes the virus in endosomes wherein acidification of
the lumen induces this refolding of the viral coat. In either case, entry into potential host cells requires
a favorable interaction between a protein on the surface of the virus and a protein on the surface of the
cell. For the purposes of gene therapy, one might either want to limit or expand the range of cells
susceptible to transduction by a gene therapy vector. To this end, many vectors have been developed in
which the endogenous viral envelope proteins have been replaced by either envelope proteins from other
viruses, or by chimeric proteins. Such chimera would consist of those parts of the viral protein
necessary for incorporation into the virion as well as sequences meant to interact with specific host cell
proteins. Viruses in which the envelope proteins have been replaced as described are referred to
aspseudotyped viruses. For example, the most popular retroviral vector for use in gene therapy trials
has been the lentivirus Simian immunodeficiency virus coated with the envelope proteins, G-
protein, from Vesicular stomatitis virus. This vector is referred to as VSV G-pseudotyped
lentivirus, and infects an almost universal set of cells. This tropism is characteristic of the VSV G-protein
with which this vector is coated. Many attempts have been made to limit the tropism of viral vectors to one
or a few host cell populations. This advance would allow for the systemic administration of a relatively
small amount of vector. The potential for off-target cell modification would be limited, and many concerns
from the medical community would be alleviated. Most attempts to limit tropism have used chimeric
envelope proteins bearingantibody fragments. These vectors show great promise for the development
of "magic bullet" gene therapies.
[edit]Replication-Competent Vectors

A replication-competent vector called ONYX-015 is used in replicating tumor cells. It was found that in the
absence of the E1B-55Kd viral protein, adenovirus caused very rapid apoptosis of infected, p53(+) cells,
and this results in dramatically reduced virus progeny and no subsequent spread. Apoptosis was mainly
the result of the ability of EIA to inactivate p300. In p53(-) cells, deletion of E1B 55kd has no consequence
in terms of apoptosis, and viral replication is similar to that of wild-type virus, resulting in massive killing of
cells.

A replication-defective vector deletes some essential genes. These deleted genes are still necessary in
the body so they are replaced with either a helper virus or a DNA molecule.
[6]

[edit]Cis and trans-acting elements

Replication-defective vectors always contain a “transfer construct”. The transfer construct carries the
gene to be transduced or “transgene”. The transfer construct also carries the sequences which are
necessary for the general functioning of the viral genome: packaging sequence, repeats for replication
and, when needed, priming of reverse transcription. These are denominated cis-acting elements, because
they need to be on the same piece of DNA as the viral genome and the gene of interest. Trans-acting
elements are viral elements, which can be encoded on a different DNA molecule. For example, the viral
structural proteins can be expressed from a different genetic element than the viral genome.
[6]

[edit]Herpes Simplex Virus

The Herpes simplex virus is a human neurotropic virus. This is mostly examined for gene transfer in
the nervous system. The wild type HSV-1 virus is able to infect neurons. Infected neurons are not rejected
by the immune system. Though the latent virus is not transcriptionally apparent, it does possess neuron
specific promoters that can continue to function normally[further explanation needed]. Antibodies to HSV-1 are
common in humans, however complications due to herpes infection are somewhat rare.[7]

[edit]Non-viral methods
Non-viral methods present certain advantages over viral methods, with simple large scale production and
low host immunogenicity being just two. Previously, low levels of transfection and expression of the
gene held non-viral methods at a disadvantage; however, recent advances in vector technology have
yielded molecules and techniques with transfection efficiencies similar to those of viruses.
[edit]Injection of Naked DNA

This is the simplest method of non-viral transfection. Clinical trials carried out of intramuscular injection of
a naked DNA plasmid have occurred with some success; however, the expression has been very low in
comparison to other methods of transfection. In addition to trials with plasmids, there have been trials with
naked PCR product, which have had similar or greater success. Cellular uptake of naked DNA is
generally inefficient. Research efforts focusing on improving the efficiency of naked DNA uptake have
yielded several novel methods, such aselectroporation, sonoporation, and the use of a "gene
gun", which shoots DNA coated gold particles into the cell using high pressure gas.[8]
[edit]Physical Methods to Enhance Delivery
[edit]Electroporation

Electroporation is a method that uses short pulses of high voltage to carry DNA across the cell
membrane. This shock is thought to cause temporary formation of pores in the cell membrane, allowing
DNA molecules to pass through. Electroporation is generally efficient and works across a broad range of
cell types. However, a high rate of cell death following electroporation has limited its use, including clinical
applications.
More recently a newer method of electroporation, termed electron-avalanche transfection, has been used
in gene therapy experiments. By using a high-voltage plasma discharge, DNA was efficiently delivered
following very short (microsecond) pulses. Compared to electroporation, the technique resulted in greatly
increased efficiency and less cellular damage.

[edit]Gene Gun

The use of particle bombardment, or the gene gun, is another physical method of DNA transfection. In
this technique, DNA is coated with gold particles and loaded into a device which generates a force to
achieve penetration of DNA/gold into the cells.

[edit]Sonoporation

Sonoporation uses ultrasonic frequencies to deliver DNA into cells. The process of acoustic cavitation
is thought to disrupt the cell membrane and allow DNA to move into cells.

[edit]Magnetofection

In a method termed magnetofection, DNA is complexed to magnetic particles, and a magnet is placed

underneath the tissue culture dish to bring DNA complexes into contact with a cell monolayer.
[edit]Chemical Methods to Enhance Delivery
[edit]Oligonucleotides

The use of synthetic oligonucleotides in gene therapy is to inactivate the genes involved in the disease
process. There are several methods by which this is achieved. One strategy uses antisense specific to
the target gene to disrupt the transcription of the faulty gene. Another uses small molecules of RNA
called siRNA to signal the cell to cleave specific unique sequences in the mRNA transcript of the faulty
gene, disrupting translation of the faulty mRNA, and therefore expression of the gene. A further strategy
uses double stranded oligodeoxynucleotides as a decoy for the transcription factors that are required to
activate the transcription of the target gene. The transcription factors bind to the decoys instead of the
promoter of the faulty gene, which reduces the transcription of the target gene, lowering expression.
Additionally, single stranded DNA oligonucleotides have been used to direct a single base change within
a mutant gene. The oligonucleotide is designed to anneal with complementarity to the target gene with
the exception of a central base, the target base, which serves as the template base for repair. This
technique is referred to as oligonucleotide mediated gene repair, targeted gene repair, or targeted
nucleotide alteration.

[edit]Lipoplexes and polyplexes

To improve the delivery of the new DNA into the cell, the DNA must be protected from damage and
(positively charged). Initially, anionic and neutral lipids were used for the construction of lipoplexes for
synthetic vectors. However, in spite of the facts that there is little toxicity associated with them, that they
are compatible with body fluids and that there was a possibility of adapting them to be tissue specific; they
are complicated and time consuming to produce so attention was turned to the cationic versions.

Cationic lipids, due to their positive charge, were first used to condense negatively charged DNA
molecules so as to facilitate the encapsulation of DNA into liposomes. Later it was found that the use of
cationic lipids significantly enhanced the stability of lipoplexes. Also as a result of their charge, cationic
liposomes interact with the cell membrane, endocytosis was widely believed as the major route by
which cells uptake lipoplexes. Endosomes are formed as the results of endocytosis, however, if genes
can not be released into cytoplasm by breaking the membrane of endosome, they will be sent to
lysosomes where all DNA will be destroyed before they could achieve their functions. It was also found
that although cationic lipids themselves could condense and encapsulate DNA into liposomes, the
transfection efficiency is very low due to the lack of ability in terms of “endosomal escaping”. However,
when helper lipids (usually electroneutral lipids, such as DOPE) were added to form lipoplexes, much
higher transfection efficiency was observed. Later on, it was figured out that certain lipids have the ability
to destabilize endosomal membranes so as to facilitate the escape of DNA from endosome, therefore
those lipids are called fusogenic lipids. Although cationic liposomes have been widely used as an
alternative for gene delivery vectors, a dose dependent toxicity of cationic lipids were also observed which
could limit their therapeutic usages.

The most common use of lipoplexes has been in gene transfer into cancer cells, where the supplied
genes have activated tumor suppressor control genes in the cell and decrease the activity of oncogenes.
Recent studies have shown lipoplexes to be useful in transfecting respiratory epithelial cells, so they
may be used for treatment of genetic respiratory diseases such as cystic fibrosis.

Complexes of polymers with DNA are called polyplexes. Most polyplexes consist of cationic polymers and
their production is regulated by ionic interactions. One large difference between the methods of action of
polyplexes and lipoplexes is that polyplexes cannot release their DNA load into the cytoplasm, so to this
end, co-transfection with endosome-lytic agents (to lyse the endosome that is made during endocytosis,
the process by which the polyplex enters the cell) such as inactivated adenovirus must occur. However,
this isn't always the case, polymers such as polyethylenimine have their own method of endosome
disruption as does chitosan and trimethylchitosan.

[edit]Dendrimers

A dendrimer is a highly branched macromolecule with a spherical shape. The surface of the particle

may be functionalized in many ways and many of the properties of the resulting construct are determined
by its surface.

In particular it is possible to construct a cationic dendrimer, i.e. one with a positive surface charge. When
in the presence of genetic material such as DNA or RNA, charge complimentarity leads to a temporary
association of the nucleic acid with the cationic dendrimer. On reaching its destination the dendrimer-
nucleic acid complex is then taken into the cell via endocytosis.

In recent years the benchmark for transfection agents has been cationic lipids. Limitations of these
competing reagents have been reported to include: the lack of ability to transfect a number of cell types,
the lack of robust active targeting capabilities, incompatibility with animal models, and toxicity. Dendrimers
offer robust covalent construction and extreme control over molecule structure, and therefore size.
Together these give compelling advantages compared to existing approaches.

Producing dendrimers has historically been a slow and expensive process consisting of numerous slow
reactions, an obstacle that severely curtailed their commercial development. The Michigan based
company Dendritic Nanotechnologies discovered a method to produce dendrimers using kinetically driven
chemistry, a process that not only reduced cost by a magnitude of three, but also cut reaction time from
over a month to several days. These new "Priostar" dendrimers can be specifically constructed to carry a
DNA or RNA payload that transfects cells at a high efficiency with little or no toxicity.

[edit]Hybrid methods
Due to every method of gene transfer having shortcomings, there have been some hybrid methods
developed that combine two or more techniques. Virosomes are one example; they
combine liposomes with an inactivated HIV or influenza virus. This has been shown to have more
efficient gene transfer in respiratory epithelial cells than either viral or liposomal methods alone. Other
methods involve mixing other viral vectors with cationic lipids or hybridising viruses.